Zebrafish model of blood-brain barrier to improve drug delivery to the brain

血脑屏障斑马鱼模型可改善药物向大脑的输送

• 批准号: 10487150
• 负责人: Michael Gottesman
• 金额: $ 46.44万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487150
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Astrocytes; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Caring; Cells; Collaborations; Consumption; Data; Drug Delivery Systems; Drug resistance; Firefly Luciferases; Fishes; Glial Fibrillary Acidic Protein; Goals; Homologous Gene; Human; Knockout Mice; Light; Luciferases; Malignant Neoplasms; Modeling; Monitor; Mus; Nature; Permeability; Play; Property; Resistance; Role; Signal Transduction; Site; Study models; Substrate Specificity; System; Time; Transgenic Organisms; Variant; Zebrafish; base; blood-brain barrier crossing; blood-brain barrier permeabilization; coelenterazine; high throughput analysis; high throughput screening; improved; inhibitor/antagonist; luciferin; mouse model; nanoluciferase; novel; prevent; promoter

Not only are ABC transporters responsible for drug resistance in cancer, but they are a major component of the blood-brain barrier (BBB) and blood-placental barrier. The three most prominent transporters at the blood-brain barrier are ABCB1, ABCC1, and ABCG2. We previously developed a murine model for analysis of ABCG2 expression at the blood-brain barrier based on the fact that luciferin is an ABCG2 substrate and its entry into the brain is prevented by transporter expression. In this model, firefly luciferase is under the expression of the GFAP promoter, leading to its expression in the astrocytes. When mice are injected with luciferin, no light signal from the brain is detected due to ABCG2 preventing luciferin from crossing the blood-brain barrier. However, when luciferin is coadministered with an ABCG2 inhibitor, it is able to cross the blood-brain barrier and react with luciferase expressed in the astrocytes to yield a light signal. Because studies of the BBB in mice are time-consuming and expensive, we are developing homologous models in the zebrafish, as components of the zebrafish BBB appear to be very similar to those of the mammalian BBB. Two transgenic zebrafish lines have been developed with either firefly luciferase or nanoLuc luciferase under the control of the GFAP promoter. Luciferin is the substrate for firefly luciferase and is transported by ABCG2, while coelenterazine is the substrate for nanoLuc and is transported by both ABCB1 and ABCG2. Thus, either model could potentially be used to study the role of transporters at the blood-brain barrier, but they could also be used to screen compounds that might increase permeability of the barrier. If zebrafish are to be considered an appropriate model for study of transporters at the blood-brain barrier, the zebrafish homologs of human transporters must be carefully characterized. Zebrafish do not have a direct homolog of human ABCB1 but instead have 2 similar variants-Abcb4 and Abcb5. Expression of these transporters in heterologous systems has enabled their detailed characterization and inhibition properties. In collaboration with Matthew Hall at NCATS, we have found that zebrafish Abcb4 is nearly identical to human ABCB1 in conferring resistance to nearly 100 known ABCB1 substrates. Zebrafish Abcb4 localizes to the BBB and other barrier and excretory sites in zebrafish. Zebrafish also have 4 homologs of human ABCG2-Abcg2a, Abcg2b, Abcg2c and Abcg2d. We have recently localized Abcg2a to the zebrafish blood-brain barrier and a detailed characterization of the substrate specificity of the transporters is underway. Preliminary data in transfected cells suggest that Abcg2a has the most similar substrate specificity to human ABCG2, but they are not identical.

ABC 转运蛋白不仅负责癌症的耐药性，而且是血脑屏障 (BBB) 和血胎盘屏障的主要组成部分。血脑屏障上三个最重要的转运蛋白是 ABCB1、ABCC1 和 ABCG2。我们之前开发了一种小鼠模型，用于分析血脑屏障处的 ABCG2 表达，基于以下事实：荧光素是 ABCG2 底物，并且转运蛋白表达会阻止其进入大脑。在该模型中，萤火虫荧光素酶在 GFAP 启动子的表达下，导致其在星形胶质细胞中表达。当给小鼠注射荧光素时，由于 ABCG2 阻止荧光素穿过血脑屏障，因此检测不到来自大脑的光信号。然而，当荧光素与 ABCG2 抑制剂共同给药时，它能够穿过血脑屏障并与星形胶质细胞中表达的荧光素酶反应，产生光信号。由于对小鼠 BBB 的研究既耗时又昂贵，因此我们正在斑马鱼中开发同源模型，因为斑马鱼 BBB 的成分似乎与哺乳动物 BBB 的成分非常相似。在 GFAP 启动子的控制下，使用萤火虫荧光素酶或 nanoLuc 荧光素酶开发了两种转基因斑马鱼品系。荧光素是萤火虫荧光素酶的底物，由 ABCG2 运输，而腔肠素是 nanoLuc 的底物，由 ABCB1 和 ABCG2 运输。因此，这两种模型都可以用于研究转运蛋白在血脑屏障中的作用，但它们也可以用于筛选可能增加屏障渗透性的化合物。如果斑马鱼被认为是研究血脑屏障转运蛋白的合适模型，则必须仔细表征人类转运蛋白的斑马鱼同源物。斑马鱼没有人类 ABCB1 的直接同源物，而是有 2 个相似的变体 - Abcb4 和 Abcb5。这些转运蛋白在异源系统中的表达使其具有详细的表征和抑制特性。通过与 NCATS 的 Matthew Hall 合作，我们发现斑马鱼 Abcb4 与人类 ABCB1 几乎相同，对近 100 种已知的 ABCB1 底物具有抗性。斑马鱼 Abcb4 定位于斑马鱼的血脑屏障和其他屏障和排泄位点。斑马鱼还具有人类 ABCG2 的 4 个同源物——Abcg2a、Abcg2b、Abcg2c 和 Abcg2d。我们最近将 Abcg2a 定位于斑马鱼血脑屏障，并且正在对转运蛋白的底物特异性进行详细表征。转染细胞的初步数据表明，Abcg2a 与人 ABCG2 具有最相似的底物特异性，但它们并不相同。